Iranian Journal of Fisheries Sciences

DOI: 10.22092/ijfs.2018.117925

Nutrient removal from aquaculture effluent using settling

ponds and filter-feeding species (Amphibalanus amphitrite

and Saccostrea cucullata): an in-situ study

Kohan A.1; Nasrolahi A.

1*; Aeinjamshid K.

2; Kiabi B.H.

1

Received: November 2017 Accepted: December 2017

Abstract

The potential application of settling ponds and two fouling filter-feeder species

Amphibalanus amphitrite and Saccostrea cucullata to bioremediate semi-intensive

shrimp farm effluent through 6 hours residence time was investigated. Settling pond

reduced total nitrogen, total phosphorus and chlorophyll a to 80.5%, 77.8% and 94.3%

of the initial concentrations (2.47±0.07 mg L-1

, 0.154±0.006 mg L-1

, and 24.44±2.02 µg

L-1

), respectively. Among S. cucullata, A. amphitrite and combination of both species,

oysters showed the highest efficiency in nutrient removal. Total nitrogen, total

phosphorus and chlorophyll a diminished respectively to 70.6%, 67.7% and 40.9% of

the initial concentrations in oyster treatments. These proportions were respectively

81.5%, 63.2% and 72.4% for ponds containing barnacles, and 69.3%, 71.2% and 44.9%

of the initial amounts in the combination of the two species treatment. Among three

different densities used for treatments, medium density of oysters (0.54 oyster per liter)

showed comparable effectiveness in nutrients and phytoplankton removal to the high

density. Lower ammonia production along with imposing less costs and effort, as well

as relatively equal ability; suggest the medium density of S. cucullata as the most

suitable choice. Our results suggest that applying settlement ponds, and particularly

with filter-feeder species such as S. cucullata, might mitigate the adverse impacts of

shrimp wastewater, including coastal eutrophication, on adjacent ecosystems.

Keywords: Aquaculture effluent, Coastal ecosystem, Filter-feeding, Persian Gulf,

Bioremediation

1-Department of Aquatic Biotechnology, Faculty of Life Sciences and Biotechnology,

Shahid Beheshti University, G. C., Evin, Tehran, 1983969411

2-Iran Shrimp Research Centre (ISRC), Iranian Fisheries Science Research Institute,

Agricultural Research Education and Extension Organization (AREEO), P.O.Box 1374,

Bushehr, Iran

*Corresponding author's Email: a_nasrolahi@sbu.ac.ir

2

Introduction

Aquaculture in general and shrimp

culture in particular has undergone a

remarkable growth during the last

decades (FAO, 2016). Increase in areas

under shrimp culture and changing

systems to semi-intensive and intensive

have resulted in the introduction of

many pollutants into the adjacent

ecosystems (Trott et al., 2004; Herbeck

et al., 2013). Organic and inorganic

nitrogen (N) and phosphorus (P)

discharging from aquaculture facilities,

could be used by and stimulate

excessive growth of autotrophic and

heterotrophic organisms (Tyrrell, 1999;

Middelburg and Nieuwenhuize, 2000)

and might cause coastal eutrophication

and marine habitat destruction (Trott

and Alongi, 2000; Herbeck et al.,

2013). To mitigate these adverse

effects, different physicochemical (e.g.,

reverse osmosis, ion exchange and

activated carbon absorption) and

biological (e.g. constructed wetlands,

biofloc technology and biofilters)

methods have been used (Liu et al.,

2007; Lin et al., 2010; Mook et al.,

2012; Song et al., 2016). Limitations of

physicochemical wastewater treatment

processes such as accumulation of

removed nutrients and pollutants in the

systems, precipitation of salts on the

membranes and biofouling problems

(Mook et al., 2012), in conjunction with

high costs of establishment and

maintenance, as well as requiring power

and complicated equipment make these

techniques less desirable in aquaculture

wastewater treatment. As limitations of

settling ponds and biological methods

are less, these techniques can be

considered economically and

environmentally more feasible,

especially in less developed countries,

to remediate aquaculture wastewaters.

The particulate matter suspending in

shrimp effluent owing to their

characteristics (e.g. high organic

proportion, and proper size) (Kinne et

al., 2001; Jackson et al., 2003; Herbeck

et al., 2013), could be efficiently

assimilated via filter-feeding process

(Erler et al., 2004; De Azevedo et al.,

2015). Many previous studies have

focused on the ability of filter-feeder

organisms, including bivalves,

crustaceans and annelids as bio-filters

of wastewaters (Jones et al., 2001;

Kinne et al., 2001; Milanese et al.,

2003; Erler et al., 2004; Giangrande et

al., 2005; Gifford et al., 2007;

Kinoshita et al., 2008; Ramos et al.,

2009; De Azevedo et al., 2015).

When selecting species to employ as

bioremediator, more appropriate

choices are sessile, native and

ubiquitous species (Gifford et al.,

2007). In the present study, barnacle,

Amphibalanus amphitrite (Darwin,

1854) and oyster, Saccostrea cucullata

(Born, 1778) as two common and

abundant fouling species residing

around the shrimp culture sites in the

northern Persian Gulf, were selected to

bio-remediate shrimp farm effluents.

Fouling filter-feeders, in addition to

having the capacity of filtering

suspended particles from the water

column (Wisely and Reid, 1978;

Cranford et al., 2011), are relatively

independent of the sediments type and

grain size of treatment ponds because

they permanently attach to hard

3

substrates. Furthermore, these species

can be collected from the wild

relatively easily. By choosing the

appropriate substrate type and placing

them in a suitable location for

settlement, a target biofouling species

can be provided (Faimali et al., 2004;

Tanyaros, 2011) and transferred to the

intended area. These characteristics, in

general, suggest that these species can

be potentially suitable options for

bioremediation of shrimp culture

effluents.

The present study aimed to

investigate the capability of settling

ponds with or without bio-filters (S.

cucullata and A. amphitrite) on N, P

and Chl-a removal from shrimp

effluent. Moreover, we tried to

determine the most appropriate

combination of the species, effective

species densities, and more efficient

effluent retention time in the treatment

ponds to achieve the highest

performance.

Materials and methods

Location

The present study was carried out in the

Shif shrimp site (SSS) adjacent to the

Bushehr Bay in the northern coasts of

the Persian Gulf (Fig. 1). The shrimp

culture areas in SSS is about 361 ha

having around 86.6 million shrimps in

stock (stocking density of

approximately 24 shrimp m-2

). The

Pacific white shrimp Litopenaeus

vannamei (Boone, 1931) is cultured in

the SSS using semi-intensive farming

system.

Figure 1: Location map of Shif shrimp site

adjacent to the Bushehr Bay, Iran.

Experimental design

To measure the efficiency of the oyster,

S. cucullata and the barnacle, A.

amphitrite in N and P removal from

shrimp farm effluent, adult specimens

of barnacles and oysters were collected

in July-August 2016. The oysters were

collected from the wild populations. To

collect the barnacles, PVC panels

(15×15 cm) were deployed at 1 m depth

for a period of two months to have

enough adult barnacles attached to

them.

Specimens were then transferred to

the treatment ponds two weeks prior to

the experiments to acclimatize to the

environmental conditions. A set of

ponds with the capacity of 500 L were

constructed closed to the effluent

channel of the SSS. Three treatments

(barnacle, oyster and a combination of

the two) with three densities for each

4

treatment (Table 1) were applied. One

pond remained without specimen and

applied as settling pond (control). Since

the size and weight of the individuals of

the two species are extremely different

(Table 1), the filtration by equal

densities could not be comparable.

Therefore, low, medium and high

densities of each species were chosen

regarding their size and weight. The

experiments were conducted in three

series and three replicates were used for

each treatment in each series (i.e. in

total nine replicates per treatment).

Water samples were taken before the

treatments and after one, three and six

hours from the beginning, to analyze

the amount of nitrite (NO2-N) and

nitrate nitrogen (NO3-N), ammonia

(NH3-N), total nitrogen, total

phosphorus and chlorophyll a.

Moreover, temperature, salinity,

dissolved oxygen, and pH were

measured at the beginning and the end

of treatments.

Table 1: Densities (low, medium and high) applied for Amphibalanus amphitrite and Saccostrea

cucullata (individual per liter) at each treatment and the mean (± SE) length and weight of

each species.

Species Weight (G) Length (cm) Low Medium High

A. mphitrite (AA) 1.20±0.31 0.88±0.26 10 20 30

S. cucullata (SC) 36.15±7.03 6.01±1.18 0.27 0.54 0.80

AA + SC - - 5+0.13 10+0.27 15+0.40

Analytical procedures

To measure Chl-a content, a certain

volume of water was filtered through

Whatman glass fiber filters (GF/F).

After extracting phytoplankton from

water sample, applied filters were

immediately frozen. The pigments

extraction was conducted using aqueous

acetone and spectrophotometry was

subsequently applied to determine the

optical density of the extract (Clesceri

et al., 1998).

Ammonia nitrogen was determined

in accordance with the oxidation

method by Parsons (2013). This

photometric technique was employed

for measurement of ammonia through

the oxidation reaction with hypochlorite

in an alkaline medium. Nitrite and

nitrate were measured using

colorimetric and cadmium reduction

methods, respectively (Clesceri et al.,

1998). Total nitrogen measurement was

carried out by oxidative digestion of

total nitrogen compounds of the shrimp

effluent sample to nitrate, and

subsequent measuring of nitrate

(Clesceri et al., 1998). All digestible

organic and inorganic forms of nitrogen

were converted to nitrate through

alkaline oxidation at temperatures of

100 to 110 °C. The ascorbic acid

method was applied to determine total

phosphorus of water samples (Clesceri

et al., 1998). Phosphorus, in this

procedure, is released from phosphorus

compounds as liberated orthophosphate,

afterwards, orthophosphate in the water

sample was measured by colorimetric

analysis.

5

Statistical analyses

The normality and homogeneity of the

data were checked using the Shapiro-

Wilk’s and Levene’s tests, respectively.

Prior to analysis, non-normal data were

transformed to meet the assumption of

normality. Statistically significant

differences among variables after one,

three and six hours of retention, with

different treatments (barnacle, oyster

and combination of the two) and

different densities of each treatment

(low, medium and high) were tested

using three-way repeated measures

ANOVA, followed by Bonferroni post-

hoc test. When the data did not meet the

assumption of sphericity (Mauchly‘s

test for sphericity), p-values were

corrected using the Greenhouse-Geisser

correction. All statistical tests were

performed using SPSS software (ver.

21).

Results

Effluent characteristics

During the course of the experiments,

average water temperature, salinity, pH

and dissolved oxygen concentration in

the shrimp farm effluent were 27.7±0.3

°C, 40.8±0.2 ppt, 7.52±0.11 and

7.27±0.61 mg L-1

, respectively.

The mean concentration of

nitrite/nitrate (NOx), total ammonia

(TAN), total nitrogen (TN), total

phosphorus (TP) and Chl-a in shrimp

farm effluent, settling ponds and

barnacle and oyster treatments at

different densities are presented in

Table 2.

Nitrite/nitrate and total ammonia

Changes in NOx concentrations, as

inorganic forms of nitrogen in shrimp

pond effluent, among three different

treatments and settling ponds were not

significantly different (p>0.05, Fig. 2A,

B, C). Different densities exhibited no

significant effect on NOx

concentrations (p>0.05). Nevertheless,

a significant reduction in NOx occurred

by increasing residence time in all

ponds (p<0.05, Fig. 2A, B, C).

Among treatments (oyster, barnacle

and the combination) no significant

difference in terms of TAN was

detected (p>0.05), although all

treatments were significantly different

from settling pond (p<0.05). Effluent

TAN concentration with a mean of

0.262±0.027 mg L-1

(Table 2) showed a

significant increase in all treatments

after 6 h (p<0.05, Fig. 2D, E, F). In

settling ponds (control), in contrast,

TAN concentration decreased after 6 h.

Total nitrogen

The amount of TN in the shrimp

effluent was initially 2.47±0.07 mg L-1

(Table 2). All treatments with different

densities and settling ponds exhibited

significant difference from shrimp

effluent (pre-treatment samples) in

terms of TN (p<0.05, Fig. 2G, H, I).

Among treatments, oyster and

combination of species showed a

significant difference in TN removal

from barnacle treatment (p<0.05).

Settling pond, oyster, barnacle and the

combination of the two species, reduced

the TN concentration to 80.5%, 70.6%,

81.5% and 69.3% of the initial

6

concentration respectively, at high

density and after 6 h.

Total phosphorus

In comparison to shrimp effluent,

settling pond as well as all treatments

reduced TP significantly (p<0.05).

Among three treatments, barnacles

showed the highest efficiency in TP

reduction compared to oyster and

combination (p<0.05). The highest

reduction in TP was observed in

barnacle ponds with the high density in

which TP content reached 63.2% of the

initial amount (i.e. 0.154±0.006 mg L-1

)

after 6 h (Fig. 2L). Oyster and

combination of the two species in the

highest density and 6 h of filtration

decreased the TP to the 67.7% and

71.2%, respectively (Fig. 2K, J). Only

medium and high densities of

treatments were significantly different

from settling ponds in TP removal

terms (p<0.05).

Chlorophyll a

Chlorophyll a concentration in all

treatments were significantly different

from the effluent Chl-a content

(p<0.05), but settling ponds did not

show any significant difference from

effluent in terms of Chl-a (p>0.05).

Oysters reduced the Chl-a content of

the effluent more effectively compared

to the other treatments (p<0.05). The

initial concentration of Chl-a in the

effluent reduced continuously with

time, being the lowest at the highest

density of oysters (40.9% of initial

concentration after 6 h, Fig. 2M). A

similar trend of Chl-a reduction with

increasing the density and filtration

time of the combination of the two

species (44.9% of initial concentration

after 6 h, Fig. 2N) and barnacles (72.4%

of initial concentration after 6 h, Fig.

2O) was observed but with a lower rate.

Settling pond by decreasing Chl-a

content to 94.3% of the initial amount

did not efficiently reduce it after 6 h.

Table 2: Concentrations of different water quality parameters (mean±SD) in the shrimp effluent

(pre-treatment sample), settling ponds and various treatments after six hours of filtration

(different letters indicating significance difference between cases). Data for one and three

hours after filtration are not presented in the table and can be followed in Fig. 2.

Treatment NOx

(mg L-1

)

Ammonia

(mg L-1

)

Total N

(mg L-1

)

Total P

(mg L-1

)

Chlorophyll a

(µg L-1

)

Effluent 0.035±0.004a

0.262±0.027a

2.47±0.07a

0.154±0.006a

24.44±2.02a

Settling pond 0.029±0.010b

0.206±0.038b

2.04±0.25b

0.129±0.017b

22.47±4.73a

Oyster-Low 0.031±0.008b

0.328±0.066c

2.11±0.27b

0.114±0.007b

14.40±6.13bc

Oyster-Med 0.025±0.008b

0.374±0.067c

1.82±0.19c

0.100±0.006c

10.73±4.89b

Oyster-High 0.027±0.007b

0.387±0.083d

1.74±0.19c

0.096±0.005c

09.53±4.80b

Comb.-Low 0.029±0.001b

0.264±0.007c

2.29±0.13b

0.113±0.005b

15.30±1.48c

Comb.-Med 0.025±0.001b

0.264±0.020c

2.12±0.08b

0.102±0.002c

10.63±1.23b

Comb.-High 0.025±0.001b

0.293±0.013d

1.89±0.09c

0.099±0.004c

12.80±1.54b

Barnacle-

Low 0.029±0.003

b 0.248±0.025

c 1.87±0.13

c 0.104±0.005

b 18.37±2.15

ac

Barnacle-

Med 0.028±0.002

b 0.257±0.030

c 1.82±0.14

c 0.099±0.006

c 18.13±2.44

ac

Barnacle-

High 0.029±0.001

b 0.260±0.034

c 1.84±0.09

c 0.085±0.006

d 16.40±2.07

c

7

Figure 2: Changes (proportions of the initial concentrations in %) in NOx (A, B, C), ammonia (D,

E, F), total nitrogen (G, H, I), total phosphorus (J, K, L) and Chlorophyll a (M, N, O), in

the experimental tanks containing oysters, combination of the two species and barnacles

respectively, during the course of experiments.

Discussion

Settling pond, in the present study,

exhibited a considerable effect in

reduction of TN, TP and Chl-a in

shrimp farm wastewater. This approach

was, however, not as effective as ponds

containing bio-filters. Nutrients were

lower in the ponds contacting barnacles,

8

oysters and combination of the two

species among which, oysters showed

the highest efficiency in nutrients and

Chl-a removal. The results showed that

TN, TP and Chl-a diminished

respectively to 70.6%, 67.7% and

40.9% of the initial concentrations in

oyster treatments. The observed higher

ability of S. cucullata in TN, TP and

Chl-a removal compared to A.

amphitrite may be partially due to the

filtration mechanism in bivalves in

which a huge part of organic and

inorganic suspended matter is removed

from the water column in the forms of

faeces and pseudo-faeces (Shumway et

al., 1985).

The capability of oysters in nutrient

removal from aquaculture wastewaters

is different among various species.

Jones and Preston (1999) indicated that

oyster (Saccostrea commercialis)

filtration, at a density of 24 individuals

per 34 L tank, resulted in a reduction of

TN, TP and Chl-a concentrations to

80%, 67% and 8% of the initial

concentrations after a period of 2 h.

Kinne et al. (2001) used the oyster

Crassostrea virginica to remediate

effluents of an intensive shrimp farm.

The authors demonstrated that this

oyster can effectively eliminate Chl-a.

Jones et al. (2001) using an integrated

treatment process, applied S.

commercialis to remediate shrimp farm

effluent. Based on the results, this

species could reduce total Kjeldahl

nitrogen, TP and Chl-a to 67.3%,

62.9% and 8.5% of the contents at the

beginning within 24 h. Crassostrea

rhizophorae and C. gigas in a study of

Ramos et al. (2009), decreased Chl-a

content to 0.0% and 17.6% of the initial

concentration in shrimp wastewater

after 6 h. In addition to filter-feeding,

oyster shells of living or dead

specimens have the capability to

remove phytoplankton and nutrients

from the water column (Kwon et al.,

2004; Caffrey et al., 2016). As well as

settling ponds, oysters can be used in

raceways to filter phytoplankton from

water (Kinne et al., 2001) and even

may show a higher efficiency than

being used in settling ponds (Jones et

al., 2002). In contrast to oysters,

barnacles did not exhibit a remarkable

performance in nutrient removal. The

potential use of barnacle, Balanus

(Amphibalanus) amphitrite for

eliminating P and N from the Salton

Sea was studied by Geraci et al. (2008).

The authors deployed hard substrates in

the water column for barnacle larval

settlement, and periodically harvested

the adult individuals grown on the

provided substrates. The results

indicated that this species did not have

enough efficiency to remove the huge

amount of N and P from this water

body.

Changes in nutrients and Chl-a

concentrations in settling pond seem to

be due to microbial activities and

physical precipitation (Erler et al.,

2004). The microbial community,

including eukaryotes and prokaryotes,

could consume a considerable part of

organic (e.g. urea, amino-acids) and

inorganic (e.g. ammonia, nitrate)

nutrients derived from shrimp culture

process (Wheeler and Kirchman, 1986;

Middelburg and Nieuwenhuize, 2000).

These microorganisms can assimilate

9

nutrients, therefore transform dissolved

forms to particles with larger sizes (i.e.

microbial biomass), which could be

assimilated through filter-feeding or

precipitated in settling ponds (Teichert-

Coddington et al., 1999; Castine et al.,

2012). In the present study,

precipitation of phytoplankton, N and P

compounds in settling ponds was not as

effective as bio-filters in terms of

nutrients and Chl-a removal. In order to

increase the efficiency of settling ponds

in nutrient removal from aquaculture

effluents, using biological methods in

conjunction with settlement ponds is

recommended (Castine et al., 2012).

Filter-feeders can be used to extract

nutrients from water rather than being

precipitated in settlement ponds floors,

through consuming a part of

bioavailable nutrients. As S. cucullata

is an edible oyster, employing this

species can be considered as a new

source of income for shrimp farmers.

Growing farm numbers and stocking

density of farms might result in the

introduction of more nutrients to the

adjacent ecosystems (Thomas et al.,

2010). In addition, at the final phase of

shrimp growth, discharging nutrients

from culture ponds would remarkably

increase (Teichert-Coddington et al.,

1999; Costanzo et al., 2004). Given

these facts, settling ponds alone may

not have required efficiency, and using

oysters as the method with the highest

efficiency should be contemplated.

Despite the reduction in TN, the

concentration of TAN increased in

settling ponds and ponds containing

oysters and barnacles. Ammonia

excretion by barnacles and oysters

(White and Walker, 1981; Jones et al.,

2002) and ammonia production through

mineralization of organic matter

(Hargreaves, 1998; Erler et al., 2007),

along with grazing phytoplankton, as

ammonia consumers, by filter-feeders

(Hargreaves, 1998), seems caused a rise

in ammonia concentration. Since

ammonia is considered as a toxic

substance for aquatic organisms

(Martinelle and Häggström, 1993;

Barbieri, 2010) and higher oyster

densities could lead to more ammonia

release, therefore, lower densities of

oysters might be a more suitable choice.

Based on the results, medium density of

oysters (0.54 oyster L-1

) could have the

relatively equal efficiency compared to

the high density (0.80 oyster L-1

).

Therefore, as the medium density of

oysters produces less TAN and lower

stocking density of oysters requires less

effort and imposes fewer costs, using

the medium density seems to be

sufficient.

In conclusion, there is a potential to

apply settling ponds with 6 h retention

time to reduce nutrients and

phytoplankton loads of shrimp effluent.

Nevertheless, to achieve higher

efficiency in nutrients and

phytoplankton removal using oyster S.

cucullata within settling ponds is

recommended.

Acknowledgements

The present study was supported by a

grant from Shahid Beheshti University.

Iran Shrimp Research Centre staff are

gratefully acknowledged for their

support and participation in the study.

10

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